The human genome harbors the genetic variations for a large number of Mendelian disorders. Many of these disorders have been localized in the genome through linkage studies, and the genes for these disorders are being isolated by different methods. The techniques currently used for isolating genes include: cDNA selection (Lovett, M., et al., Proc. Natl. Acad. Sci. USA, 88:9628-32 (1991)), exon trapping (Duyk, G. M., et al., Proc. Nail. Acad. Sci. USA, 87:8995-9 (1990)), CpG island identification (Estivill, X. and Williamson, R., Nucleic Acids Res., 15: 1415-25 (1987)), hybridization using genomic fragments as probes against cDNA libraries (Rommerns, et al., Science, 245:1059-80 (1989)), cloning and sequencing of genomic DNA followed by computer analysis of the possible coding regions (Wilson, R., et al., Nature, 368:32-38 (1994)), Alu-splice PCR (Fuentes, J. J., et al., Hum. Genet. 101:346-50 (1997)), and Alu-promoter PCR (Jendraschak, E. and Kaminski, W. E., Genomics, 50:53-60 (1998)).
These techniques have several limitations. For example, many require analyzing large numbers of subclones to yield meaningful results. Both cDNA selection and hybridization using genomic fragments depend upon gene expression patterns using cDNA or mRNA libraries. Exon trapping requires specialized vectors and cell culture materials; whilst cDNA selection results only in an enrichment of expressed sequences from a specific RNA source followed by much time and effort to determine the origin of the selected cDNAs. Alu-splice PCR also has limitations in that it can identify only a few putative exons out of a larger number of true exons, even in a YAC clone. Because none of these methods permit the isolation of all the genes in a given region, normally several of the above methods are used in conjunction to complement one another, thereby achieving more complete results.
Furthermore, these methods are most usually only applied to DNA regions included in vectors such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), plasmids, and cosmids. They cannot be applied directly to whole genomic DNA for isolating a majority of the exons of genes contained in the genome. A method for isolating the majority of the flanking regions to a signal sequence, such as the 3′ or the 5′ splice junction or the promoters, present at numerous locations in a genome with a consensus sequence, would be very a advantageous in a variety of genetic studies for discovering and treating major illnesses.
In essence, current methods for specifically amplifying exons present in an fir; unknown genomic DNA are limited in their abilities. The isolation of only exon sequences from a gene will be advantageous for a variety of applications including comparative analysis between individuals. Attempts have been made to use the above methods to accomplish this purpose using genomic DNA fragments cloned into vectors.
For example, the Alu-splice PCR method attempts to isolate exon-containing fragments from cloned genomic DNA. This method utilizes the consensus sequence of splice junctions linked to a restriction enzyme recognition sequence as one primer and the consensus sequence of Alu repeat elements as the other primer to amplify any potential exon sequence that may be present between these primer binding sites in a cloned YAC DNA. The results of this method are poor for many reasons. For example, in one study, from a total of 128 colonies picked, only ten contained putative exons. Furthermore, out of the few genes present in the two YACs analyzed, none of the nine exons present in one of the genes was isolated. Further still, most of the exons from among the five new genes that possibly existed in these YACs were not isolated except for one or two exons. From among the ten putative exon sequences isolated, six were shorter than 350 nucleotides. As the authors of this study agree, not all genes in a given sample will be identified by Alu-splice PCR, and not all the exons within a given gene will be identified by Alu-splice PCR. There are at least two reasons that explain this outcome: 1) the paucity of conveniently placed Alu repetitive elements; and 2) the limiting factor of specificity of the 5′ and 3′ splice-site primers; in the best of cases, primer specificity is only eight nucleotides. These inadequate results, even with a relatively short template DNA (YAC) compared to genomic DNA, indicate that this method is not applicable to isolate, in multiplex fashion, the exons of many genes from whole genomic DNA.